A BODIPY/pyrene-based chemodosimetric fluorescent chemosensor for selective sensing of hydrazine in the gas and aqueous solution state and its imaging in living cells

Abstract

A BODIPY-based pyrenebutyrate-linked (BPB) chromogenic and fluorogenic probe was synthesized and characterized for the specific detection of hydrazine. In the presence of hydrazine, BODIPY-based pyrenebutyrate was selectively deprotected, producing switch off meso-phenoxyBODIPY along with a color change from yellow to brown, allowing colorimetric detection of hydrazine by the naked eye. Selectivity experiments proved BPB has excellent selectivity to hydrazine over other environmentally abundant ions and common amine-containing species. Probe BPB was also successfully applied in vapor hydrazine detection into a solid state over other interfering volatile analytes. Furthermore, the probe BPB coated with silica gel TLC plates could act as a visual and fluorimetric probe for hydrazine vapor detection. The probe (BPB) has been shown to detect hydrazine up to 1.87 μM at pH 7.4. DFT and TDDFT calculations were performed in order to demonstrate the sensing mechanism and the electronic properties of the probe and hydrazinolysis product. BPB can also be used for the detection of hydrazine in Vero cells without appreciable interference from other biologically abundant analytes.

---

Introduction

The development of selective fluorescent chemosensors to provide signals in response to chemically, biologically and environmentally important small molecules is an interesting area of contemporary research in supramolecular chemistry.¹ In this regard, the development of a new strategy for molecular sensors involves the application of analyte triggered selective transformations of chemosensor derivatives to yield signaling on–off fluorochromes based on versatile dyes of BODIPY, rhodamine, and fluorescein.^2–4

Hydrazine is an important chemical reagent such as highly reactive base and strong reducing agent used in organic group transformation. For instance, in industries, hydrazine is employed as a corrosion inhibitor for boilers due to strong reducing power to scavenge oxygen, pharmaceutical intermediate, catalyst, emulsifier and antioxidant agent acts as a preservative in nuclear and electrical power plants.⁵ Hydrazine also plays important roles in the preparation of pesticides, emulsifiers, drug intermediates, photography chemicals, and dyes in various chemical industries.⁶ It is famous as a high-energy fuel in missiles, satellites and rocket-propulsion systems due to its flammable and detonable characteristics.⁷ In contrast to its usefulness, carcinogenic and toxic effects of hydrazine potentially lead to serious environmental contamination and serious health risks during its manufacture, use, transport and disposal procedures. Hydrazine vapor and its water solutions, however, are highly toxic to humans and animals causes severe damage to the liver, lungs, kidneys, central nervous system and other documented adverse systemic health effects includes irritation of eyes, nose, and throat, temporary blindness, dizziness, nausea, pulmonary edema, coma, and blood abnormalities and non-specific chronic illnesses.⁸ Therefore, hydrazine has been classified as a carcinogenic substance by the U.S. Environmental Protection Agency (EPA) with allowable threshold limit value (TLV) of 10 ppb,^9,10 which provides an incentive to research for new analytical approaches capable of determining trace level of hydrazine are always required.

Various traditional analytical techniques for the detection of hydrazine are available, including electrochemical analysis¹¹ and chromatography,¹² including gas chromatography,¹³ HPLC,¹⁴ coulometry,¹⁵ potentiometry,¹⁶ titrimetry,¹⁷ capillary electrophoresis¹⁸ and so on.¹⁹ Spectrophotometry using colored derivatives, such as p-dimethylaminobenzaldehyde²⁰ and chlorosalicylaldehyde,²¹ is also used to detect hydrazine. However, these methods are not only complex and time consuming, but also impractical for in vivo hydrazine analysis because of their post-mortem processing and destruction of tissues and cell contents. Among several detection strategies, fluorescent techniques are extremely attractive due to their high sensitivity, low cost, easy implementation, and real-time detection.^22,23 Till now, only a few fluorescent chemodosimeters for hydrazine have been reported, and almost all of them were designed based on the deprotection or chemical transformation of a protecting group by a specific deprotecting agent or analyte.^24–28 For example, the fluorescent sensing system developed by Chang et al.,²⁹ showed the selective deprotection of levulinated coumarin in presence of hydrazine to a chemosensor to date in DMSO–water. The sensing system reported by Peng et al.,³⁰ showed a ratiometric hydrazine-selective NIR probe based on cyanine dye via deprotection of acetyl group in aqueous-organic solvent. Therefore, it still remains a challenge to develop effective hybrid fluorescent probe with suitable reactive zone can act as good chemodosimetric chemosensor for the recognition of molecular species, though such systems are limited in the literature in case of hydrazine sensing. Furthermore, fewer sensors have been applied to vapor sensing.^31,32 Some of these current probes could only be utilized in low pH (pH < 5) conditions^33,34 which would limit their application in physiological condition. Thus, developing a new fluorescence method of monitoring hydrazine in living cells or sensing vapor hydrazine remains a significant challenge.

Our research group has been involved in the synthesis of various chemosensors-based BODIPY–pyrene conjugates for the selective recognition of ions and molecules.^35,36 We recently reported the selective detection of anions and CNS active alkaloids using BODIPY, pyrene and carbazole based chemosensor. Therefore, it is of prime interest to develop BODIPY–pyrene-based hybrid reactive molecular systems to provide better sensitivity and selectivity toward sensing of toxic molecules. With this in mind, in the present paper, the synthesis and characterization of BODIPY–pyrene conjugates of chemodosimeter, BPB and their selective recognition behavior toward hydrazine have been reported. Here two fluorophores were connected in the chemodosimeter BPB, because these two fluorophores has high quantum yields, high extinction coefficient, and both their excitation and emission wavelengths are in the visible region.⁴⁰ Considering that hydrazine shows strong nucleophilicity towards ester groups, the specific chemical reaction between dosimeter molecule BPB and hydrazine yielded the products of hydrazone and phenolate ion of BODIPY respectively. After the hydrazinolysis, the original phenolic form of the hidden probe BODIPY becomes free again, which affected the intramolecular electron density distribution of BODIPY in the meso position, therefore resulting in ICT-induced absorption and an efficient photoinduced electron transfer (PET) quenching fluorescence responses to hydrazine in solution. To the best of our knowledge, this is the first example of a chemodosimetric detection of solution as well as vapour hydrazine using BODIPY–pyrene hybrid fluorophores, which shows ON–OFF behavior due to both PET and ICT processes in the BODIPY–pyrene conjugate after hydrazinolysis. These conjugate was also demonstrated first successful intracellular application as potential live-cell fluorescence imaging agents upon treatment with hydrazine.

Experimental section

General information and materials

Unless otherwise mentioned, materials were obtained from commercial suppliers and were used without further purification. ¹H and ¹³C NMR spectra were recorded on a Brucker 400 MHz instrument. For NMR spectra, DMSO-d₆ was used as solvent using TMS as an internal standard. Chemical shifts are expressed in δ ppm units. Mass spectra were carried out using a Waters QTOF Micro YA 263 mass spectrometer. UV-visible and fluorescence spectra measurements were performed on a JASCO V530 and a Photon Technology International (PTI-LPS-220B) spectrofluorimeter respectively. Elemental analysis of the compounds was carried out on Perkin-Elmer 2400 series CHNS/O Analyzer. Chemicals and solvents used for the synthesis of receptor were purchased from Sigma Aldrich Chemical Co. (USA) and used without further purification. Salts of different cations, anions and amines were purchased from Spectrochem Pvt Ltd (India). The following abbreviations are used to describe spin multiplicities in ¹H NMR spectra: s = singlet; d = doublet; t = triplet; m = multiplet.

General method of UV-vis and fluorescence titration

For UV-vis and fluorescence titrations, stock solution of sensor BPB was prepared (c = 1 × 10⁻⁵ ML⁻¹) in H₂O–DMSO (3 : 7, v/v) solution (10 mM HEPES buffer, pH 7.4). The solution of the guest cations, anions and amines in the order of 2 × 10⁻⁴ ML⁻¹ was also prepared in H₂O–DMSO (3 : 7, v/v) solution (10 mM HEPES buffer, pH 7.4). The test solution of sensor BPB was prepared by proper dilution method. The spectra of these solutions were recorded by means of UV-vis and the fluorescence methods. All the solvents were purchased from local suppliers and were distilled by standard procedure before use.

Cell culture

Vero cell (Vero 76, ATCC no CRL-1587) lines were prepared from continuous culture in Dulbecco's modified Eagle's medium (DMEM, Sigma Chemical Co., St. Louis, MO) supplemented with 10% fetal bovine serum (Invitrogen), penicillin (100 μg mL⁻¹), and streptomycin (100 μg mL⁻¹). The Vero 76 were obtained from the American Type Culture Collection (Rockville, MD) and maintained in DMEM containing 10% (v/v) fetal bovine serum and antibiotics in a CO₂ incubator. Cells were initially propagated in 75 cm² polystyrene, filter-capped tissue culture flask in an atmosphere of 5% CO₂ and 95% air at 37 °C in CO₂ incubator. When the cells reached the logarithmic phase, the cell density was adjusted to 1.0 × 10⁵ per well in culture media. The cells were then used to inoculate in a glass bottom dish, with 1.0 mL (1.0 × 10⁴ cells) of cell suspension in each dish. After cell adhesion, culture medium was removed. The cell layer was rinsed twice with phosphate buffered saline (PBS), and then treated according to the experimental need.

Cellular imaging methodology

For fluorescence imaging studies Vero cells, 1 × 10⁴ cells in 1000 μL of medium, were seeded on sterile 35 mm Petri dish, glass bottom culture dish (ibidi GmbH, Germany), and incubated at 37 °C in a CO₂ incubator for 10 hours. Then cells were washed with 500 μL DMEM followed by incubation with final concentration of 1.0 × 10⁻⁶ M BPB probe dissolved in 500 μL DMEM at 37 °C for 1 h in a CO₂ incubator and observed under an Olympus IX71 microscope. Images analyzed by Image-pro plus (version 6.3) with excitation at nearby 495 nm FITC filter, and emit at nearby 519 nm. The cells were again washed thrice with phosphate buffered saline PBS (pH 7.4) to remove any free BPB probe and incubated in PBS containing NH₂–NH₂ to a final concentrations of 1.0 × 10⁻⁴ M, incubated for 30 min followed by washing with PBS three times to remove excess NH₂–NH₂ outside the cells and images were captured.

Synthesis of compound (BPB)

Compound (2) (100 mg, 0.294 mmol), γ-oxo-1-pyrenebutyric acid (89 mg, 0.294 mmol), EDC (56.4 mg, 0.294 mmol) and DMAP (35.9 mg, 0.294 mmol) in dichloromethane (30 mL) was stirred at room temperature for 24 h. Then water was added to the solution and extracted with CH₂Cl₂. The organic layer was washed with water and dried over MgSO₄. After concentration of solvent, the residue was purified via chromatography with silica gel (10% hexane in CHCl₃) to afford BPBas a reddish brown solid (yield 81%). M.P = 233–235 °C. ¹H NMR (d₆-DMSO, 400 MHz) δ (ppm): 8.926 (d, 1H, Ar–H, J = 9.6 Hz), 8.681 (d, 1H, Ar–H, J = 8.0 Hz), 8.415 (t, 3H, Ar–H, J = 7.8 Hz), 8.352 (d, 2H, Ar–H, J = 8.0 Hz), 8.292 (t, 1H, Ar–H, J = 7.0 Hz), 8.174 (t, 1H, Ar–H, J = 7.6 Hz), 7.431 (dd, 4H, Ar–H, J = 6.8 Hz, J = 6.4 Hz), 6.181 (s, 2H), 3.767 (t, 2H, pyrene –COCH₂–, J = 6.2 Hz), 3.174 (t, 2H, ester –OCOCH₂–, J = 6.0 Hz), 2.546 (s, 6H), 1.301 (s, 6H). ¹³C NMR (d₆-DMSO, 100 MHz) δ (ppm): 14.58, 31.11, 72.46, 76.37, 88.77, 106.11, 121.95, 122.94, 123.84, 124.05, 124.67, 124.93, 125.15, 125.47, 126.34, 127.15, 127.24, 127.57, 127.64, 128.09, 129.68, 130.25, 130.67, 130.86, 132.02, 134.67, 139.35, 144.95, 162.75, 184.51, 206.71. TOF MS ES+, m/z = 647.0407 [BPB + Na]⁺; calculated, 647.4727 anal. cald for C₃₉H₃₁N₂O₃BF₂; C, 75; H, 5; N, 4.486 found: C, 75.12; H, 4.92; N, 4.46%.

Results and discussion

The chemodosimeter BPB was synthesized by going through the steps given in Scheme 1. Initially, BODIPY part was synthesized by the condensation of 4-hydroxybenzaldehyde with 2,4-dimethylpyrrole according to a published procedure.³⁶ The final dosimeter molecule, BPB has been synthesized in a single step by reacting BODIPY with γ-oxo-1-pyrenebutyric acid in presence of coupling agent EDC/DMAP in dichloromethane at room temperature. All the synthesized molecules were well characterized by various analytical and spectral techniques.

Scheme 1 Schematic representation of synthesis of BPB. Solid-state fluorescence images of 2, γ-oxo-1-pyrenebutyric acid and BPB under 366 nm ambient light irradiation.

To understand the reactive properties of BPB towards hydrazine, studies were carried out by emission, absorption, and ¹H NMR spectroscopy, and the species of recognition has been addressed by mass spectrometry and computations based on the density functional theory (DFT). The detection of hydrazine vapor by silica gel plate is also demonstrated. The pyridomethene–BF₂ complex dye is selected as a fluorescent transducer due to its high fluorescence intensity both in solution and in solid state.

In order to find the suitability of BPB to react hydrazine in aqueous buffer solution at physiological pH, fluorescence titrations were carried out in different solvent mixture at room temperature. As we know, nucleophilic substitution reactions are better conducted in organic solutions. Therefore, we firstly investigated the emission properties of probe BPB in pure DMSO. We found that the addition of 2 equiv. of hydrazine could result in an immediate drop of fluorescence intensity at both 456 and 516 nm respectively. As shown in Fig. S7 in the ESI,† the fluorescence spectra changes in different aqueous HEPES buffer in H₂O–DMSO solution are similar to those in pure DMSO but the drop of fluorescence intensity is slower than that in pure DMSO. We speculate this phenomenon was probably due to the hydrogen bond between hydrazine and water in aqueous solution that weakened the nucleophilicity of hydrazine to BPB and then decreased the reaction speed. Finally, the fluorescence signalling behaviour of BODIPY–pyrene-based probe BPB was investigated in an optimized aqueous solution HEPES buffer (pH 7.4, 10 mM) in H₂O–DMSO (3 : 7, v/v) at room temperature.

The probe BPB exhibit very strong emission bands at 456 and 516 nm due to pyrene and BODIPY moieties respectively when excited at 356 nm in 3 : 7 H₂O–DMSO mixture at pH 7.4 to have an effective HEPES buffer concentration of 10 mM. By contrast, upon gradual addition of hydrazine to the probe BPB, the emission intensity bands at 456 and 516 nm sharply decreases along with the solution color change from bright green to colorless under a UV lamp. When the concentration of hydrazine reached 2 equiv., a maximal fluorescence decrease (up to a 15.5-fold decrease at 516 nm) was observed (Fig. 1). A plot of fluorescence intensity as a function of added [Hydrazine]/[BPB] mole ratio (Fig. 1b) shows a stoichiometry of 1 : 1 between the probe and hydrazine and the intensity goes to lowest value at ≥1 equiv.

Fig. 1 (a) Fluorescence spectra (excitation at 356 nm) of sensor BPB (c = 1 × 10⁻⁵ ML⁻¹) in H₂O–DMSO (3 : 7, v/v) solution (10 mM HEPES buffer, pH 7.4) in the presence of 0–2.0 equiv. of hydrazine and (b) the relative fluorescence intensity (I/I₀) as a function of [N₂H₄]/[BPB] mole ratio.

The observed fluorescence off may be viewed as follows. We speculate that the signalling change was due to the selective deprotection of the 4-oxo-4-pyrenylbutyrate of probe BPB by hydrazine and generate hidden phenolate unit of BODIPY (3). The product generated by the reaction of probe BPB with hydrazine is confirmed to be compound 3 and 4 (Scheme 2) by mass spectrometry analysis (Fig. S8 in the ESI†). Our previous work^36–39 has demonstrated that a phenoxy substituent or any electron donating group at the meso (8) position of the BODIPY core plays crucial role for photoinduced-electron-transfer (PET)-based quenching of the emission. To confirm the validity of the proposed sensing mechanism, a solution of probe BPB was analyzed by ¹H NMR in the absence and presence of hydrazine. The protons of BODIPY moieties moved up field when hydrazine was added, which obviously suggested that the probe BPB totally converted to oxonium with the removal of the 4-oxo-4-pyrylbutyrate (Scheme 2). Thus, the detection limit of the probe for hydrazine was calculated (2σ/slope) to be 1.87 μM (Fig. S9 in the ESI†).

Scheme 2 Hydrazine-selective signaling mechanism by BPB.

To prove the selectivity of probe BPB, the competitive fluorescence titrations were carried out in the presence of common anions and cations, redox molecules and amines. As shown in Fig. 2, upon addition of 30 equiv. of anions (the common ions included F⁻, Cl⁻, Br⁻, I⁻, CN⁻, N₃⁻, PO₄³⁻, NO₃⁻, S²⁻, SO₄²⁻, HSO₄⁻ as their sodium salts), and amines (NH₂OH, ethylene diamine and NH₃) exhibited almost no changes in emission behaviour.

Fig. 2 (a) UV-vis spectra and (b) fluorescence spectra of BPB (c = 1 × 10⁻⁵ M) in H₂O–DMSO (3 : 7, v/v) solution (10 mM HEPES buffer, pH 7.4) in the presence of hydrazine (2 equivalents) and other representative cations, anions and organic bases (30 equivalents each).

Meanwhile, the commonly encountered cations [Cu²⁺, Hg²⁺, Zn²⁺, Cd²⁺, Mg²⁺, Al³⁺, Fe³⁺ and Ag⁺ (as their chloride salts)] did not cause any change to the fluorescence of probe BPB (Fig. 2). The results indicate that the probe BPB has high selectivity for hydrazine over other species. This may be attributed to the unique chemical reaction between 4-oxo-4-pyrylbutyrate and hydrazine.

Fluorescence spectra were also recorded for the titration of probe BPB against hydrazine in the presence of 30 equiv. of common anions and cations, redox anions and amines. None of these analyte significantly affects the emission intensity of BPB upon the addition of hydrazine, and the titration profile is similar to that obtained for simple hydrazine titration (Fig. S10 in the ESI†). Therefore, it can be concluded that probe BPB selectively reacts hydrazine even in the presence of other analytes.

In order to ascertain the reactivity of hydrazine by probe BPB, absorption titrations were carried out by adding varying concentrations of hydrazine to a fixed concentration (c = 1 × 10⁻⁵ M) of BPB. We found that the addition of trace amounts of hydrazine causes the absorption and fluorescence signal to change rapidly, which is very important for real-time detection. Therefore, the following titration experiments were carried out after gradual addition of hydrazine.

The absorption spectra of free BPB in aqueous DMSO (3 : 7 v/v) HEPES buffer (pH 7.4) exhibited different bands from 250 to 550 nm. The absorbance of 284, 356, 438 and 501 nm bands is found to decrease gradually upon addition of hydrazine (Fig. 3a), indicating the interaction of hydrazine with the reactive groups such as ester and carbonyl present in the probe BPB.

Fig. 3 (a) Changes in the UV/vis absorption spectra of sensor BPB (c = 1 × 10⁻⁵ M) in H₂O–DMSO (3 : 7, v/v) solution (10 mM HEPES buffer, pH 7.4) in the presence of 0–2.0 equiv. of hydrazine and (b) the photograph of visible color (top) under ambient light and visual fluorescence color (bottom) changes of sensor BPB with various relevant analytes in H₂O–DMSO (3 : 7, v/v) solution under a hand-held UV lamp (366 nm): BPB only; (1) NH₂OH; (2) NH₂(CH₂)₂NH₂; (3) NH₃; (4) F⁻; (5) Cl⁻; (6) Br⁻; (7) I⁻; (8) CN⁻; (9) N₃⁻; (10) PO₄³⁻; (11) N₂H₄; (12) NO₃⁻; (13) S²⁻; (14) SO₄²⁻; (15) HSO₄⁻; (16) Cu²⁺; (17) Hg²⁺; (18) Zn²⁺; (19) Cd²⁺; (20) Mg²⁺; (21) Al³⁺; (22) Fe³⁺ and (23) Ag⁺.

The absorption behavior changed the color of the solution from light green to orange, allowing colorimetric detection of hydrazine by the naked eye. The color of the fluorescence also changed clearly from green to colorless. However the absorption titration carried out with all the other common cations and anions as well as redox anions showed no significant change, indicating their noninteractive nature with BPB. Even the different amines do not react to BPB at room temperature.

We speculate that the 4-oxo-4-pyrenylbutyrate moiety was selected as the reaction site on the fluorophore, based on a specific reactivity between BPB and hydrazine for the production of pyrene and BODIPY deivatives. Hydrazine was detected by hydrazinolysis of the oxo-pyrene-butarate moiety of BPB (Scheme 2).

This reaction generated meso-phenoxyBODIPY (3) which exhibited its characteristic chromogenic and fluorescent behaviors.

The reaction of BPB with N₂H₄ was illustrated by involving two steps, at first the nucleophilic addition–elimination to the carbonyl group at the 4-position of the 4-oxobutyrate group and then the second nucleophilic addition–elimination to the ester carbonyl with subsequent amide ring formation leading to cleavage of the ester function to release the meso-phenoxyBODIPY moiety. This is consistent with the hydrazine-promoted deprotection of the levulinate group.²⁹ We suggest that the nonfluorescent of the hydrazinolysis product, which resulted from the efficient PET quenching of the excited state of the BODIPY moiety, but the ICT process we mentioned above is the only reason that gives rise to large changes in the absorption and emission spectra.

To confirm the validity of the proposed sensing mechanism, a solution of probe BPB was analyzed by ¹H NMR in the absence and presence of hydrazine. By ¹H NMR spectroscopy, resonances for the phenyl parts of BODIPY moiety of probe BPB were observed at 7.46 and 7.41 ppm. After hydrazine was added, the aromatic protons H_a and H_b in probe BPB moved up field to 7.12 and 6.93 ppm respectively, which obviously suggested that the probe BPB totally converted to oxonium with the removal of the oxobutyrate group and matched the characteristic chemical shift for the aromatic protons H_a and H_b in meso-phenoxyBODIPY. In addition, the resonances of the reaction product pyrene derivative of tetrahydropyridazinone were also observed (Fig. 4). The characteristic methylene protons of H_c (pyrene –COCH₂–) and H_d (ester –OCOCH₂–) at 3.76 and 3.17 ppm respectively in probe BPB disappeared in probe [BPB + N₂H₄], and the methylene protons of H_c (pyrene –COCH₂–) and H_d (ester –OCOCH₂–) at 2.57 and 2.71 ppm respectively in pyrene derivative of tetrahydropyridazinone were clearly found, indicating the clean cleavage of the ester bond. The UV-vis and fluorescence spectrum of probe BPB in the presence of hydrazine was also almost identical to that of the proposed deprotection product of phenoxy BODIPY itself (Fig. S11 in the ESI†). To further understand the mechanism of probe BPB with hydrazine, ESI-MS was used to test the solutions containing BPB and 5 equiv. hydrazine, and the peak at 341.00 instead of 647.0407 proved that the ester group had been removed and generation of new peak at 299.2 corresponds to pyrene derivative of tetrahydropyridazinone.

Fig. 4 Partial ¹H NMR (400 MHz) spectra of BPB only and [BPB + N₂H₄]. [BPB] = 4 × 10⁻³ M, [Hydrazine] = 1 × 10⁻² M in D₂O/DMSO-d₆ (3 : 7, v/v).

To understand the spectroscopic properties of the new BPB probe in the solid state, the solid-state absorption and emission spectra, the thin films were prepared by evaporation of 500 μM dyes in CH₂Cl₂ onto a silica plates.

The visual color of BPB is bright red in solid state, and its fluorescence spectrum is shown in Fig. 5a. After dipping into a CH₂Cl₂ solution of BPB followed by drying, the silica gel plate looks orange, yet emits a red color under UV light (366 nm) (Fig. 5c). Solid state sensors are convenient to use and much easier to carry around for practical usage. As anticipated, the new dyes in the solid state display a red-shift (Δδ = 85 nm) in the emission profiles relative to in dichloromethane. In great contrast to most BODIPY dyes, which barely exhibit fluorescence in their solid-state due to the small Stokes-shifts.^41,42 By sharp contrast, our new BPB dyes have large stokes shifts, which bodes well for the solid-state emission. Indeed, BPB display intense reddish-orange emission in the solid state (Fig. 5a).

Fig. 5 Fluorescence spectra of (a) BPB and (b) compound-2 in the solid state (film). The excitation wavelength was 356 nm and 370 nm for BPB and compound-2 respectively. The inset shows the solid-state fluorescence images of BPB and 2 under 366 nm UV-light irradiation. (c) Photographs showing the colors of BPB in solution and solid state on TLC plate strips under ambient (left) and UV light (right).

By contrast, compound-2 shows solid-state crimson red emission (Fig. 5b). Aprahamian and co-workers showed⁴³ that the solid-state fluorescent of boron difluoride (BF₂) complexes depends on several parameters: (1) the planarity of the molecule, (2) the dipole moment, (3) the number of π–π interactions between neighboring dyes, and (4) the peripheral decoration of BODIPY by bulky substituents (preferably in meso position) in order to avoid significant aggregation caused quenching (ACQ).^44,45 So this luminescent compound show interesting optical properties both in solution and the solid-state.^46–48

The fluorescence spectra of BPB in different solvents were measured as shown in Fig. 6b. The emission displays a significant solvatochromic shift to longer wavelength upon decreasing the solvent polarity. The polarity of the system from acetonitrile to toluene/hexane led to a slight to moderate red shift of the absorption/emission maxima.

Fig. 6 Normalized (a) absorption and (b) emission spectra of BPB recorded in different solvents [λ_exc = 356 nm].

The shift of λ_em increases from 516 nm in acetonitrile up to 528 nm in a hexane nonpolar solvent. It indicates that the dipole moment of BPB after the photo-excitation was not so much increased as there is no ICT present in the molecule.

To get insight into the optical response of probe BPB to hydrazine, the probe BPB and the corresponding products obtained from the reaction between probe BPB and hydrazine were examined by density function theory (DFT) and time-dependent density function theory (TDDFT) calculations at the TDB3LYP/6-311G(d,p)//B3LYP/6-31G(d,p) level of the Guassian 09 program.⁴⁹

The optimized geometries and calculated electron distributions in the frontier molecular orbitals HOMO and LUMO of BPB and the two fragmented products of BODIPY moiety and pyrene-hydrazone moiety are shown in Fig. 7. In particular, for the probe BPB, both the HOMO and LUMO are mostly localized at the BODIPY moiety. As well as planar BODIPY ring fragment is almost perpendicular to the adjacent phenyl part with dihedral angle 89.5°. The major chromophore BODIPY adopts a nonplanar conformation with other part of the probe, and hence the ICT character of the sensor is only very weak as result no absorption and emission shift happen. Only the formation of the local emission (LE) bands at 501 and 453 nm for BODIPY and pyrene moieties were observed. The formation of the LE emission bands at 501 and 453 nm in the hydrazine products are likely due to the nonplanar and nonconjugated character of the meso position.

Fig. 7 Calculated (B3LYP/6-311G(d,p)//B3LYP/6-31G(d,p) level) geometry and HOMO–LUMO energy levels and the interfacial plots of the orbitals for sensor BPB, 3 and 4 in solvent phase.

In addition, we also performed time-dependent density function theory (TD-DFT) calculations for the reactant as well as both the product also. The vertical transitions i.e., the calculated λ_max, main orbital transition, and oscillator strength (f) are listed in Table S1 in the ESI.† In the case of the BPB probe, TDDFT calculations provided absorption band at ∼427, ∼393 and ∼255 nm belonging to the S₀ → S₁ (f = 0.5740), S₀ → S₄ (f = 0.5401) and S₀ → S₂₈ (f = 0.4601) energy states respectively (Tables S1 and S2 in the ESI†). The main contributing transitions are S₀ → S₁ (∼98.0%) and S₀ → S₄ (∼94.6%), which corresponds to energy states arises from HOMO → LUMO and HOMO−1 → LUMO+1 respectively. These values are consistent with the absorbance bands at 438 and 356 nm obtained experimentally. In particular for BODIPY product, the MO's are majorly localized on phenoxide conjugated BODIPY unit, and the main contribution transition comes from HOMO → LUMO (100%) with a greater oscillator strength (f = 0.3563) provided a calculated absorption band at ∼577 nm, which also mainly corresponds to the absorbance band at 501 nm obtained experimentally. Similarly, the calculated band at ∼386 nm of pyrene-hydrazone is assigned to the vertical major transition of HOMO → LUMO (95.6%) (Fig. 7) that results from a π → π* transition within the pyrene fragment, which mainly corresponds to the experimentally observed absorbance band at 356 nm. Notably, there is no ICT between the BODIPY and pyrene moieties, which could be the main reason for the Local Emissions (LE) of the two fluorophores.

In order to show the practical utility of BPB for the detection of hydrazine, we further tested whether probe BPB could be applicable in the detection of gas-state hydrazine that is universal during industrial processes among other vaporizable organic compounds. To make the detection experiments easy to perform and practical, test strips were used. Prior to detection, test strips were prepared by sinking the filter paper with probe BPB (5.0 mM) of CH₂Cl₂ solution and then dried. The probe-loaded test strips were covered on the top of jars that contained different hydrazine solution concentrations (blank, 0.1%, 0.5%, 1%, 5%, 10%, 20%, 25%, 30%, 40% and 50% in water) for 10 min at room temperature before it was ready to observe. The fluorescence color of the strips gradually faded, which proved that the fluorescence intensity of the probe on the test strips were highly dependent on the concentration of hydrazine in aqueous solution and easy to distinguish with the naked eye (Fig. 8).

Fig. 8 Fluorescence color changes of probe BPB (5.0 mM) coated filter paper after exposure to different concentrations of hydrazine aqueous solution. The fluorescence color changes were collected using a hand-held UV lamp (366 nm).

Thus, one highlight of this probe is that BPB could identify hydrazine vapor even with a concentration as low as 0.5%, which is considerably more sensitive than the recently developed hydrazine probes.^31,32 It is noteworthy that the probe has high potential applications in hydrazine detection. Hydrazine in gaseous form often threatens human life, so our designed probe has more application potential.

We also explored opportunities for probe BPB to analyze hydrazine in aqueous solution for practical applications. Because hydrazine has carcinogenic properties, and has been widely used in a variety of industrial processes, hydrazine detection in aqueous samples is of interest.

Prior to living cell imaging, probe BPB was used to detect hydrazine in tap water and distilled water. An aliquot of hydrazine was added to water and the recoveries obtained by BPB signals were compared in tap water and distilled water (Fig. 9). The analysis of hydrazine in both solutions agreed well at hydrazine concentrations up to 2.0 × 10⁻⁴ M. The results show that probe BPB can detect hydrazine in real water samples quantitatively.

Fig. 9 Fluorescence detection of hydrazine in distilled water and tap water by BPB. [BPB] = 1.0 × 10⁻⁵ M, [Hydrazine] = from 0 to 2.0 × 10⁻⁴ M in H₂O–DMSO (3 : 7, v/v) solution (10 mM HEPES buffer, pH 7.4) [λ_exc = 356 nm].

The favorable features of the probe BPB absorption and emission in the visible region, a significant fluorescence turn-off signal, high sensitivity, high selectivity and functioning well at physiological pH due to the interacting properties of probe BPB with hydrazine.

These desirable characters prompted us to evaluate the ability of the probe to image hydrazine in living cells. Prior to investigating the suitability of the probe BPB for imaging hydrazine in living cells, it is necessary to evaluate its cytotoxicity.

The standard MTT assays suggest that the probe BPB has low cytotoxicity to the living cells (Fig. 10a). Now the stage was set for cell imaging of hydrazine. The living cells were treated with the probe BPB in the absence or presence of hydrazine. Vero cells incubated with BPB (10 μM) for 20 min at 37 °C in PBS buffer with 0.5% DMSO showed strong intracellular fluorescence as shown in Fig. 10b(B). By contrast, cells pre-loaded with the probe BPB and further incubated with hydrazine for further 10 min displayed switch-off fluorescence (Fig. 10b) inside Vero cells, as observed earlier in solution studies. The fluorescence microscopic analysis strongly suggested that hydrazine could readily cross the membrane barrier of the Vero cells, and rapidly sense intracellular probe BPB by altering the intrinsic fluorescence of BPB, after hydrazinolysis. It is significant to mention here that bright field images of treated cells did not reveal any gross morphological changes, which suggested that Vero cells were viable. These findings open up the avenue for future in vivo biomedical applications of the sensor. Thus, BPB can be a suitable fluorescence chemosensing probe for hydrazine detection in biological systems.

Fig. 10 (a) MTT assay to determine the cytotoxic effect of probe BPB and BPB –N₂H₄ on Vero 76 cells. (b) Fluorescence microscopic images in Vero 76 cells: (A) bright field image of the cells, pretreated with probe BPB, (B) only probe BPB at 1.0 × 10⁻⁶ M concentration, (C) bright field image of the cells treated with N₂H₄ at concentration 1.0 × 10⁻⁴ M, (D) cells treated with probe at concentration 1.0 × 10⁻⁶ M, N₂H₄ at 1.0 × 10⁻⁴ M, and switch-off fluorescence signal is detected. All images were acquired with a 40× objective lens. Scale bar represents 20 μm.

Conclusion

A new chemosignaling probe system based on the BODIPY–pyrenebutyrate-linked (BPB) has been synthesized and characterized. The probe could selectively react with hydrazine through deprotection of pyrenebutyrate, resulting in an ON–OFF fluorescence signal change at 516 and 456 nm respectively in a 30% aqueous DMSO solution. The selectivity and sensitivity was demonstrated on the basis of fluorescence, absorption, and ¹H NMR spectroscopy, ESI mass spectrometry, and visual fluorescent color changes. The significant changes in fluorescence color can be observed by the naked eye as well as in the solid state. It is noteworthy that probe could identify hydrazine vapor even with a concentration as low as 0.5% within a few minutes at 25 °C when silica gel plate soaked with BPB. The limit of detection is 1.87 μM. The structural, electronic, and emission properties of the BPB and its reaction products, have been demonstrated using DFT computational calculations. The TDDFT calculations reveal that the switch off fluorescence behavior of 3 is mainly due to the conjugation of electron donating group at the meso (8) position of the BODIPY core. Additionally, probe BPB could be used to visualize hydrazine in living cells. This sensor has high potential as a useful tool for investigating the roles of hydrazine in biological and pathological processes.

Acknowledgements

We thank the DST-New Delhi [Project file no. SR/S₁/OC-44/2012] for financial support. SKM and SM thanks to the UGC and BP thanks CSIR, New Delhi for a fellowship.

Notes and references

1. X. Su and I. Aprahamian, Chem. Soc. Rev., 2014, 43, 1963–1981.
2. M. E. Jun, B. Roy and K. H. Ahn, Chem. Commun., 2011, 47, 7583–7601.
3. J. Du, M. Hu, J. Fan and X. Peng, Chem. Soc. Rev., 2012, 41, 4511–4535.
4. Y. Yang, Q. Zhao, W. Feng and F. Li, Chem. Rev., 2013, 113, 192–270.
5. S. D. Zelnick, D. R. Mattie and P. C. Stepaniak, Aviat., Space Environ. Med., 2003, 74, 1285–1291.
6. A. Umar, M. M. Rahman, S. H. Kim and Y. B. Hahn, Chem. Commun., 2008, 166–168.
7. H. W. Schiessl, Kirk-Othmer Encyclopedia of Chemical Technology, John Wiley& Sons, Incorporation, NJ, USA, 2000, pp. 562–607.
8. American Conference of Governmental Industrial Hygienists, Documentation of Threshold Limit Values and Biological Exposure Indices, ACGIH, Cincinnati, OH, 1999.
9. G. Choudhary and H. Hansen, Chemosphere, 1998, 37, 801–843.
10. U.S. Environmental Protection Agency (EPA), Integrated Risk Information System (IRIS) on Hydrazine/Hydrazine Sulfate,National Center for Environmental Assessment, Office of Research and Development, Washington, DC, 1999.
11. C. Batchelor-McAuley, C. E. Banks, A. O. Simm, T. G. J. Jones and R. G. Compton, Analyst, 2006, 131, 106–110.
12. D. P. Elder, D. Snodin and A. Teasdale, J. Pharm. Biomed. Anal., 2011, 54, 900–910.
13. M. Sun, L. Bai and D. Q. Lui, J. Pharm. Biomed. Anal., 2009, 49, 529–533.
14. H. Bhutani, S. Singh, S. Vir, K. K. Bhutani, R. Kumar, A. K. Chakraborti and K. C. Jindal, J. Pharm. Biomed. Anal., 2007, 43, 1213–1220.
15. E. C. Olson, Anal. Chem., 1960, 32, 1545–1547.
16. J. R. Stetter, K. F. Blurton, A. M. Valentine and K. A. Tellefsen, J. Electrochem. Soc., 1978, 125, 1804–1807.
17. H. E. Malone, Anal. Chem., 1961, 33, 575–577.
18. J. Liu, W. Zhou, T. You, F. Li, E. Wang and S. Dong, Anal. Chem., 1996, 68, 3350–3353.
19. J. Wang and Z. Lu, Electroanalysis, 1989, 1, 517–521.
20. M. George, K. S. Nagaraja and N. Balasubramanian, Talanta, 2008, 75, 27–31.
21. X. Chen, Y. Xiang, Z. Li and A. Tong, Anal. Chim. Acta, 2008, 625, 41–46.
22. D. T. Quang and J. S. Kim, Chem. Rev., 2010, 110, 6280–6303.
23. A. P. Demchenko, Advanced Fluorescence Reporters in Chemistry and Biology I, Springer, New York, 2010, 8.
24. G. E. Collins and S. L. Rose-Pehrsson, Analyst, 1994, 119, 1907–1913.
25. Y. D. Lin and T. J. Chow, RSC Adv., 2013, 3, 17924–17929.
26. M. H. Lee, B. Yoon, J. S. Kim and J. L. Sessler, Chem. Sci., 2013, 4, 4121–4126.
27. S. Goswami, S. Das, K. Aich, B. Pakhira, S. Panja, S. K. Mukherjee and S. Sarkar, Org. Lett., 2013, 15, 5412–5415.
28. M. D. Sun, J. Guo, Q. B. Yang, N. Xiao and Y. X. Li, J. Mater. Chem. B, 2014, 2, 1846–1851.
29. M. G. Choi, J. Hwang, J. O. Moon, J. Sung and S.-K. Chang, Org. Lett., 2011, 13, 5260–5263.
30. C. Hu, W. Sun, J. F. Cao, P. Gao, J. Y. Wang, J. L. Fan, F. L. Song, S. G. Sun and X. J. Peng, Org. Lett., 2013, 15, 4022–4025.
31. L. Cui, Z. Peng, C. Ji, J. Huang, D. Huang, J. Ma, S. Zhang, X. Qian and Y. Xu, Chem. Commun., 2014, 50, 1485–1487.
32. L. Xiao, J. Tu, S. Sun, Z. Pei, Y. Pei, Y. Pang and Y. Xu, RSC Adv., 2014, 4, 41807–41811.
33. J. Fan, W. Sun, M. Hu, J. Cao, G. Cheng, H. Dong, K. Song, Y. Liu, S. Sun and X. Peng, Chem. Commun., 2012, 48, 8117–8119.
34. M. G. Choi, J. O. Moon, J. Bae, J. W. Lee and S. K. Chang, Org. Biomol. Chem., 2013, 11, 2961–2965.
35. A. K. Mahapatra, S. Mondal, K. Maiti, S. K. Manna, R. Maji, S. Mandal, S. Goswami, D. Mondal, C. K. Quah and H.-K. Fun, RSC Adv., 2014, 4, 56605–56614.
36. A. K. Mahapatra, R. Maji, K. Maiti, S. S. Adhikary, C. D. Mukhopadhyay and D. Mandal, Analyst, 2014, 139, 309–315.
37. Q. Li, Y. Guo and S. Shao, Analyst, 2012, 137, 4497–4501.
38. G. Meng, S. Velayudham, A. Smith, R. Luck and H. Liu, Macromolecules, 2009, 42, 1995–2001.
39. J. O. Huh, Y. Do and M. H. Lee, Organometallics, 2008, 27, 1022–1025.
40. N. Boens, V. Leen and W. Dehaen, Chem. Soc. Rev., 2012, 41, 1130–1172.
41. J. Araneda, E. Piers, B. Heyne, M. Parvez and R. McDonald, Angew. Chem., Int. Ed., 2011, 50, 12214–12217.
42. D. Zhao, G. Li, D. Wu, X. Qin, P. Neuhaus, Y. Cheng, C. Yang, Z. Lu, X. Pu, C. Long and J. You, Angew. Chem., Int. Ed., 2013, 52, 13676–13680.
43. Y. Yang, X. Su, C. N. Carrol and I. Aprahamian, Chem. Sci., 2012, 3, 610–613.
44. H. Lu, Q. Wang, L. Gai, Z. Li, Y. Deng, X. Xiao, G. Lai and Z. Shen, Chem.–Eur. J., 2012, 18, 7852–7861.
45. L. Gai, H. Lu, B. Zou, G. Lai, Z. Shen and Z. Li, RSC Adv., 2012, 2, 8840–8846.
46. J. Massue, D. Frath, G. Ulrich, P. Retailleau and R. Ziessel, Org. Lett., 2012, 14, 230–233.
47. J. Massue, P. Retailleau, G. Ulrich and R. Ziessel, New J. Chem., 2013, 37, 1224–1230.
48. J. Massue, D. Frath, P. Retailleau, G. Ulrich and R. Ziessel, Chem.–Eur. J., 2013, 19, 5375–5386.
49. M. J. Frisch, G. W. Trucks, H. B. Schlegel, G. E. Scuseria, M. A. Robb, J. R. Cheeseman, G. Scalmani, V. Barone, B. Mennucci, G. A. Petersson, H. Nakatsuji, M. Caricato, X. Li, H. P. Hratchian, A. F. Izmaylov, J. Bloino, G. Zheng, J. L. Sonnenberg, M. Hada, M. Ehara, K. Toyota, R. Fukuda, J. Hasegawa, M. Ishida, T. Nakajima, Y. Honda, O. Kitao, H. Nakai, T. Vreven, J. A. Montgomery Jr, J. E. Peralta, F. Ogliaro, M. Bearpark, J. J. Heyd, E. Brothers, K. N. Kudin, V. N. Staroverov, R. Kobayashi, J. Normand, K. Raghavachari, A. Rendell, J. C. Burant, S. S. Iyengar, J. Tomasi, M. Cossi, N. Rega, J. M. Millam, M. Klene, J. E. Knox, J. B. Cross, V. Bakken, C. Adamo, J. Jaramillo, R. Gomperts, R. E. Stratmann, O. Yazyev, A. J. Austin, R. Cammi, C. Pomelli, J. W. Ochterski, R. L. Martin, K. Morokuma, V. G. Zakrzewski, G. A. Voth, P. Salvador, J. J. Dannenberg, S. Dapprich, A. D. Daniels, Ö. Farkas, J. B. Foresman, J. V. Ortiz, J. Cioslowski and D. J. Fox, Gaussian 09, Revision D.01, Gaussian, Inc., Wallingford, CT, 2009.

---

Footnote

† Electronic supplementary information (ESI) available. See DOI: 10.1039/c5ra10198k

---